Reagent holder, and kits containing same

ABSTRACT

A holder for reagents, such as may be used for transporting the reagents and for carrying out processing operations on biological samples with the reagents. The holders typically hold reagents for amplifying polynucleotides extracted from the samples. The holder comprises a connecting member; a process tube affixed to the connecting member and having an aperture located in the connecting member; at least one socket, located in the connecting member, and configured to accept a pipette tip; two or more reagent tubes disposed on the underside of the connecting member, each having an inlet aperture located in the connecting member; and one or more receptacles, located in the connecting member and each being configured to receive a reagent tube. Also described are reagent tubes configured with stellated shaped patterns, on their bottom interior surfaces, configured to facilitate complete or near-complete withdrawal of fluid from the tube, via a pipette tip.

CLAIM OF PRIORITY

This application claims the benefit of priority of U.S. provisionalpatent application Ser. No. 60/959,437, filed Jul. 13, 2007 which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The technology described herein generally relates to holders forreagents, such as may be used for transporting the reagents and forcarrying out processing operations with the reagents. The technologymore particularly relates to holders that hold reagents for preparingbiological samples for amplifying and detecting polynucleotidesextracted from the samples.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. For example, many diagnostic analyses can onlybe done with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations, and often there is just one in any given urban area. Thismeans that most hospitals are required to send out samples for analysesto these locations, thereby incurring shipping costs and transportationdelays, and possibly even sample loss or mishandling.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate or make efficient asmany of these as possible. In one key step, a biological sample, onceextracted from a patient, must be put in a form suitable for aprocessing and detection regime that typically involves using PCR toamplify a vector of interest. Once amplified, the presence or absence ofthe vector in the sample needs to be determined unambiguously. Preparingsamples for PCR is currently a time-consuming and labor intensive step,though not one requiring specialist skills, and could usefully beautomated. By contrast, steps such as PCR and nucleotide detection havecustomarily only been within the compass of specially trainedindividuals having access to specialist equipment.

Sample preparation is labor intensive in part because of the number ofreagents required, and the need for multiple liquid transfer (e.g.,pipetting) operations. Furthermore, the reagents required are ofsufficient variety that they typically require different handling fromone another and are available from different vendors.

There is therefore a need for a method and apparatus of carrying outsample preparation on samples, such as in parallel, with or without PCRand detection on the prepared biological samples, and so that logisticalinconveniences of reagent handling are reduced.

The discussion of the background herein is included to explain thecontext of the inventions described herein. This is not to be taken asan admission that any of the material referred to was published, known,or part of the common general knowledge as at the priority date of anyof the claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

A reagent holder comprising: a connecting member having an upper sideand, opposed to the upper side, an underside; a process tube affixed tothe connecting member and having an aperture located in the connectingmember; at least one socket, located in the connecting member, thesocket configured to accept a pipette tip; two or more reagent tubesdisposed on the underside of the connecting member, each of the reagenttubes having an inlet aperture located in the connecting member; and oneor more receptacles, located in the connecting member, wherein the oneor more receptacles are each configured to receive a reagent tubeinserted from the upper side of the connecting member.

A kit comprising: a first pouch containing one or more of the holdersdescribed herein; and a second pouch, having an inert atmosphere inside,and one or more tubes containing lyophilized PCR reagents.

A unitized reagent holder, comprising: a strip, to which is attached: asingle process tube; one or more containers, each of which holding areagent selected from the group consisting of: a sample preparationreagent, PCR reagents for a first analyte, and one or more liquidreagents; and one or more sockets configured to hold one or more pipettetips.

A reagent holder comprising: a process tube; one or more pipette tips;and one or more reagent tubes, wherein the one or more reagent tubescontain, respectively, sufficient quantities of one or more reagents forcarrying out extraction of polynucleotides from a sample, and whereinthe process tube, the one or more pipette tips, and the one or morereagent tubes are each joined to a single connecting member.

A reagent tube, comprising: a stellated pattern of cutouts or ridgescentered at the bottom of the interior surface of the tube.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an first exemplary embodiment of a reagent holderhaving a pipette sheath, in perspective view (FIG. 1A) and undersideview (FIG. 1B).

FIG. 2 shows an exemplary embodiment of a reagent holder not having apipette sheath, in perspective view.

FIGS. 3A-3C show a second exemplary embodiment of a reagent holderhaving a pipette sheath, in perspective view (FIG. 3A) andcross-sectional view (FIG. 3B), and exploded view (FIG. 3C).

FIGS. 4A and 4B show a stellated feature on the interior of a reagenttube, in cross-sectional (FIG. 4A) and plan (FIG. 4B) view.

FIG. 5 shows a sequence of pipetting operations in conjunction with areagent tube having a stellated feature.

FIG. 6 shows embodiments of a laminated layer.

FIG. 7 shows a sequence of pipetting operations in conjunction with alaminated layer.

FIGS. 8A-8D shows an exemplary kit containing holders and reagents.

FIG. 9 shows a rack for holding one or more holders.

FIG. 10 shows a process tube heating unit, as used in conjunction with arack.

FIG. 11 shows a schematic of an automated apparatus configured to carryout sample preparation using a holder as described herein.

Like reference numerals in the various drawings indicate like elements.

DETAILED DESCRIPTION

Described herein are reagent holders for holding and transportingreagents for various purposes, in particular sample preparation in aclinical context. The holder permits snapping in of one or more tubes ofanalyte specific reagents (ASR's) and/or sample preparation reagents,and carrying out liquid dispensing processes, associated with samplepreparation, that minimize cross-sample contamination but permitmultiple PCR preparations to be performed from a single clinical sample.The holders are configured for use in an automated preparatory apparatusthat can carry out sample preparation on samples in more than one holdersimultaneously.

The reagent holders as described herein find particular application toanalyzing any nucleic acid containing sample for any purpose, includingbut not limited to genetic testing, and clinical testing for variousinfectious diseases in humans.

In various embodiments, preparation of a PCR-ready sample for use insubsequent diagnosis, can include one or more of the following steps:contacting a neutralized polynucleotide sample with a PCR reagentmixture comprising a polymerase enzyme and a plurality of nucleotides(in some embodiments, the PCR reagent mixture can further include apositive control plasmid and a fluorogenic hybridization probe selectivefor at least a portion of the plasmid). In some embodiments, the PCRreagent mixture can be in the form of one or more lyophilized pellets,as stored in a container on the holder, and the method can furtherinclude reconstituting the PCR pellet with liquid to create a PCRreagent mixture solution. The holder herein provides in a self-containedmanner, all of the reagents required to prepare a PCR-ready sample, or,when delivered to a user in kit form, contains in conjunction with otherpackages all of the required reagents. Suitable reagents, and protocolsfor using the same in DNA and RNA extractions can be found in,respectively, copending application Ser. Nos. 12/172,208, and12/172,214, both filed Jul. 11, 2008 and incorporated herein byreference.

The holders herein are also configured for use by an apparatus thatcarries out automated sample preparation, for example, on multiplesamples simultaneously. An exemplary form of such an apparatus isfurther described herein, and can also be found described in U.S.provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007,incorporated herein by reference in its entirety.

Holder

FIGS. 1A and 1B show views of an exemplary holder 501 as furtherdescribed herein. FIG. 2 shows a plan view of another exemplary holder502, as further described herein. FIG. 3A shows an exemplary holder 503in perspective view, and FIG. 3B shows the same holder incross-sectional view. FIG. 3C shows an exploded view of the same holderas in FIGS. 3A and 3B. All of these exemplary holders, as well as othersconsistent with the written description herein though not shown asspecific embodiments, are now described.

The exemplary holders shown in FIGS. 1A, 1B, 2, 3A, 3B, and 3C can eachbe referred to as a “unitized disposable strip”, or a “unitized strip”,because they are intended to be used as a single unit that is configuredto hold all of the reagents and receptacles necessary to perform asample preparation, and because they are laid out in a strip format. Itis consistent with the description herein, though, that other geometricarrangements of the various receptacles are contemplated, so that thedescription is not limited to a linear, or strip, arrangement, but caninclude a circular or grid arrangement.

Some of the reagents contained in the holder are provided as liquids,and others may be provided as solids. In some embodiments, a differenttype of container or tube is used to store liquids from those that storethe solids.

The holder can be disposable, such as intended for a single use,following which it is discarded.

The holder is typically made of a plastic such as polypropylene. Theplastic is such that it has some flexibility to facilitate placementinto a rack, as further described herein. The plastic is typicallyrigid, however, so that the holder will not significantly sag or flexunder its own weight and will not easily deform during routine handlingand transport, and thus will not permit reagents to leak out from it.

The holder comprises a connecting member 510 having one or morecharacteristics as follows. Connecting member 510 serves to connectvarious components of the holder together. Connecting member 510 has anupper side 512 and, opposed to the upper side, an underside 514. In FIG.1B, a view of underside 514 is shown, having various struts 597connecting a rim of the connecting member with variously the sockets,process tube, and reagent tubes. Struts 597 are optional, and may beomitted all or in part, or may be substituted by, in all or in part,other pieces that keep the holder together.

The holder is configured to comprise: a process tube 520 affixed to theconnecting member and having an aperture 522 located in the connectingmember; at least one socket 530, located in the connecting member, thesocket configured to accept a disposable pipette tip 580; two or morereagent tubes 540 disposed on the underside of the connecting member,each of the reagent tubes having an inlet aperture 542 located in theconnecting member; and one or more receptacles 550, located in theconnecting member, wherein the one or more receptacles are eachconfigured to receive a complementary container such as a reagent tube(not shown) inserted from the upper side 512 of the connecting member.

The holder is typically such that the connecting member, process tube,and the two or more reagent tubes are made from a single piece, such asa piece of polypropylene.

The holder is also typically such that at least the process tube, andthe two or more reagent tubes are translucent.

The one or more receptacles 550 are configured to accept reagent tubesthat contain, respectively, sufficient quantities of one or morereagents typically in solid form, such as in lyophilized form, forcarrying out extraction of nucleic acid from a sample that is associatedwith the holder. The receptacles can be all of the same size and shape,or may be of different sizes and shapes from one another. Receptacles550 are shown as having open bottoms, but are not limited to suchtopologies, and may be closed other than the inlet 552 in the upper sideof connecting member 510. Preferably the receptacles 550 are configuredto accept commonly used containers in the field of laboratory analysis,or containers suitably configured for use with the holder herein. Thecontainers are typically stored separately from the holders tofacilitate sample handling, since solid reagents normally requiredifferent storage conditions from liquid reagents. In particular manysolid reagents may be extremely moisture sensitive.

The snapped-in reagent tubes containing different reagents may be ofdifferent colors, or color-coded for easy identification by the user.For example they may be made of different color material, such as tintedplastic, or may have some kind of identifying tag on them, such as acolor stripe or dot. They may also have a label printed on the side,and/or may have an identifier such as a barcode on the sealing layer onthe top.

The containers 554 received by the receptacles 550 may alternatively bean integrated part of the holder and may be the same type of containeras the waste chamber and/or the reagent tube(s), or may be differenttherefrom.

In one embodiment, the containers 554 containing lyophilized reagents,disposed in the receptacles 550 (shown, e.g., in FIGS. 3A and 3C), are0.3 ml tubes that have been further configured to have a star pattern(see FIGS. 4A and 4B) on their respective bottom interior surfaces. Thisis so that when a fluid has been added to the lyophilized reagents(which are dry in the initial package), a pipette tip can be bottomedout in the tube and still be able to withdraw almost the entire fluidfrom the tube, as shown in FIG. 5, during the process of nucleic acidextraction. The design of the star-pattern is further describedelsewhere herein.

The reagent tubes, such as containing the lyophilized reagents, can besealed across their tops by a metal foil, such as an aluminum foil, withno plastic lining layer, as further described herein.

The embodiments 501, 502, and 503 are shown configured with a wastechamber 560, having an inlet aperture 562 in the upper side of theconnecting member. Waste chamber 560 is optional and, in embodimentswhere it is present, is configured to receive spent liquid reagents. Inother embodiments, where it is not present, spent liquid reagents can betransferred to and disposed of at a location outside of the holder, suchas, for example, a sample tube that contained the original sample whosecontents are being analyzed. Waste chamber 560 is shown as part of anassembly comprising additionally two or more reagent tubes 540. It wouldbe understood that such an arrangement is done for convenience, e.g., ofmanufacture; other locations of the waste chamber are possible, as areembodiments in which the waste chamber is adjacent a reagent tube, butnot connected to it other than via the connecting member.

The holder is typically such that the connecting member, process tube,the two or more reagent tubes, and the waste chamber (if present) aremade from a single piece, made from a material such as polypropylene.

The embodiments 501 and 503 are shown having a pipette sheath 570. Thisis an optional component of the holders described herein. It may bepermanently or removably affixed to connecting member 510, or may beformed, e.g., moulded, as a part of a single piece assembly for theholder. For example, exploded view of holder 503 in FIG. 3C showslug-like attachments 574 on the upper surface of a removable pipettesheath 570 that engage with complementary recessed portions or holes inthe underside 514 of connecting member 510. Other configurations ofattachment are possible. Pipette sheath 570 is typically configured tosurround the at least one socket and a tip and lower portion of apipette tip when the pipette tip is stationed in the at least onesocket. In some embodiments, the at least one socket comprises foursockets. In some embodiments the at least one socket comprises two,three, five, or six sockets.

Pipette sheath 570 typically is configured to have a bottom 576 and awalled portion 578 disposed between the bottom and the connectingmember. Pipette sheath 570 may additionally and optionally have one ormore cut-out portions 572 in the wall 578, or in the bottom 576. Suchcutouts provide ventilation for the pipette tips and also reduce thetotal amount of material used in manufacture of the holder. Embodiment503 has a pipette sheath with no such cutouts. In embodiment 501, such acutout is shown as an isosceles triangle in the upper portion of thesheath; a similar shaped cutout may be found at a corresponding positionin the opposite side of the sheath, obscured from view in FIG. 1A. Othercutouts could have other triangular forms, circular, oval, square,rectangular, or other polygonal or irregular shapes, and be several,such as many, in number. The wall 578 of pipette sheath 570 may alsohave a mesh or frame like structure having fenestrations or interstices.In embodiments having a pipette sheath, a purpose of the sheath is tocatch drips from used pipette tips, and thereby to prevent cross-samplecontamination, from use of one holder to another in a similar location,and/or to any supporting rack in which the holder is situated.Typically, then, the bottom 576 is solid and bowl-shaped (concave) sothat drips are retained within it. An embodiment such as 502, having nopipette sheath, could utilize, e.g., a drip tray or a drainage outlet,suitably placed beneath pipette tips located in the one or more sockets,for the same purpose. In addition to catching drips, the pipette tipsheath prevents or inhibits the tips of other reagent holders—such asthose that are situated adjacent to the one in question in a rack asfurther described herein—from touching each other when the tips arepicked up and/or dropped off before or after some liquid processingstep. Contact between tips in adjacent holders is generally not intendedby, for example, an automated dispensing head that controls sampleprocessing on holders in parallel, but the pipette tips being long caneasily touch a tip in a nearby strip if the angle when dropping off ofthe tip deviates slightly from vertical.

The holders of embodiments 501, 502, and 503, all have a connectingmember that is configured so that the at least one socket, the one ormore receptacles, and the respective apertures of the process tube, andthe two or more reagent tubes, are all arranged linearly with respect toone another (i.e., their midpoints lie on the same axis). However, theholders herein are not limited to particular configurations ofreceptacles, waste chamber, process tube, sockets, and reagent tubes.For example, a holder may be made shorter, if some apertures arestaggered with respect to one another and occupy ‘off-axis’ positions.The various receptacles, etc., also do not need to occupy the samepositions with respect to one another as is shown in FIGS. 3A and 3B,wherein the process tube is disposed approximately near the middle ofthe holder, liquid reagents are stored in receptacles mounted on oneside of the process tube, and receptacles holding solid reagents aremounted on the other side of the process tube. Thus, in FIGS. 1A, 1B,and 2, the process tube is on one end of the connecting member, and thepipette sheath is at the other end, adjacent to, in an interiorposition, a waste chamber and two or more reagent tubes. Still otherdispositions are possible, such as mounting the process tube on one endof the holder, mounting the process tube adjacent the pipette tips andpipette tip sheath (as further described herein), and mounting the wastetube adjacent the process tube. It would be understood that alternativeconfigurations of the various parts of the holder give rise only tovariations of form and can be accommodated within other variations ofthe apparatus as described, including but not limited to alternativeinstruction sets for a liquid dispensing pipette head, heater assembly,and magnetic separator, as further described herein.

Process tube 520 can also be a snap-in tube, rather than being part ofan integrated piece. Process tube 520 is typically used for variousmixing and reacting processes that occur during sample preparation. Forexample, cell lysis can occur in process tube 520, as can extraction ofnucleic acids. Process tube 520 is then advantageously positioned in alocation that minimizes, overall, pipette head moving operationsinvolved with transferring liquids to process tube 520.

Reagent tubes 540 are typically configured to hold liquid reagents, oneper tube. For example, in embodiments 501, 502, and 503, three reagenttubes are shown, containing respectively wash buffer, release buffer,and neutralization buffer, each of which is used in a sample preparationprotocol.

Reagent tubes 540 that hold liquids or liquid reagents can be sealedwith a laminate structure 598. The laminate structure typically has aheat seal layer, a plastic layer such as a layer of polypropylene, and alayer of metal such as aluminum foil, wherein the heat seal layer isadjacent the one or more reagent tubes. The additional plastic film thatis used in a laminate for receptacles that contain liquid reagents istypically to prevent liquid from contacting the aluminum.

Two embodiments of a laminate structure, differing in their layerstructures, are shown in FIG. 6. In both embodiments, the heat seallayer 602, for example made of a laquer or other such polymer with a lowmelting point, is at the bottom, adjacent to the top of the holder, whenso applied. The plastic layer 604 is typically on top of the heat seallayer, and is typically made of polypropylene, having a thickness in therange 10-50 microns. The metal layer 608 is typically on top of theplastic layer and may be a layer of Al foil bonded to the plastic layerwith a layer of adhesive 606, as in the first embodiment in FIG. 6, ormay be a layer of metal that is evaporated or sputtered into placedirectly on to the plastic layer. Exemplary thicknesses for therespective layers are shown in FIG. 6, where it is to be understood thatvariations of up to a factor of 2 in thickness are consistent with thetechnology herein. In particular, the aluminum foil is 0.1-15 micronsthick, and the polymer layer is 15-25 microns thick in one embodiment.In another embodiment, the aluminum is 0.1-1 microns thick, and thepolymer layer is 25-30 microns thick.

The laminates deployed herein make longer term storage easier becausethe holder includes the presence of sealed lyophilized reagents as wellas liquids sealed in close proximity, which is normally hard to achieve.

In one embodiment, the tops of the reagent tubes have beveled edges sothat when an aluminum foil is heat bonded to the top, the plastic meltdoes not extend beyond the rim of the tube. This is advantageousbecause, if the plastic melt reduces the inner diameter of the tube, itwill cause interference with the pipette tip during operation. In otherembodiments, a raised flat portion 599 facilitates application andremoval of laminate 598. Raised surface 599, on the upper side of theconnecting member, and surrounding the inlet apertures to the reagenttubes and, optionally, the waste chamber, is an optional feature of theholder.

The manner in which liquid is pipetted out is such that a pipette tippiercing through the foil rips through without creating a seal aroundthe pipette tip, as in FIG. 7. Such a seal around the tip duringpipetting would be disadvantageous because a certain amount of air flowis desirable for the pipetting operation. In this instance, a seal isnot created because the laminate structure causes the pierced foil tostay in the position initially adopted when it is pierced. The upperfive panels in FIG. 7 illustrate the pipetting of a reagent out from areagent tube sealed with a laminate as further described herein. At A,the pipette tip is positioned approximately centrally above the reagenttube that contains reagent 707. At B, the pipette tip is lowered,usually controllably lowered, into the reagent tube, and in so doingpierces the foil 598. The exploded view of this area shows the edge ofthe pierced laminate to be in contact with the pipette tip at the widestportion at which it penetrates the reagent tube. At C, the pipette tipis withdrawn slightly, maintaining the tip within the bulk of thereagent 707. The exploded view shows that the pierced foil has retainedthe configuration that it adopted when it was pierced and the pipettetip descended to its deepest position within the reagent tube. At D, thepipette tip sucks up reagent 707, possibly altering its height as moreand more older people undergo such tests. At E, the pipette tip isremoved entirely from the reagent tube.

The materials of the various tubes and chambers may be configured tohave at least an interior surface smoothness and surface coating toreduce binding of DNA and other macromolecules thereto. Binding of DNAis unwanted because of the reduced sensitivity that is likely to resultin subsequent detection and analysis of the DNA that is not trapped onthe surface of the holder.

The process tube also may have a low binding surface, and allowsmagnetic beads to slide up and down the inside wall easily withoutsticking to it. Moreover, it has a hydrophobic surface coating enablinglow stiction of fluid and hence low binding of nucleic acids and othermolecules.

In some embodiments, the holder comprises a registration member such asa mechanical key. Typically such a key is part of the connecting member510. A mechanical key ensures that the holder is accepted by acomplementary member in, for example, a supporting rack or a receivingbay of an apparatus that controls pipetting operations on reagents inthe holder. A mechanical key is normally a particular-shaped cut-outthat matches a corresponding cutout or protrusion in a receivingapparatus. Thus, embodiment 501 has a mechanical key 592 that comprisesa pair of rectangular-shaped cut-outs on one end of the connectingmember. This feature as shown additionally provides for a tab by which auser may gain a suitable purchase when inserting and removing the holderinto a rack or another apparatus. Embodiments 501 and 502 also have amechanical key 590 at the other end of connecting member 510. Key 590 isan angled cutout that eases insertion of the holder into a rack, as wellas ensures a good registration therein when abutting a complementaryangled cut out in a recessed area configured to receive the holder.Other variations of a mechanical key are, of course, consistent with thedescription herein: for example, curved cutouts, or various combinationsof notches or protrusions all would facilitate secure registration ofthe holder.

In some embodiments, not shown in FIG. 1A, 1B, 2, or 3A-C, the holderfurther comprises an identifier affixed to the connecting member. Theidentifier may be a label, such as a writable label, a bar-code, a2-dimensional bar-code, or an RFID tag. The identifier can be, e.g., forthe purpose of revealing quickly what combination of reagents is presentin the holder and, thus, for what type of sample preparation protocol itis intended. The identifier may also indicate the batch from which theholder was made, for quality control or record-keeping purposes. Theidentifier may also permit a user to match a particular holder with aparticular sample.

It should also be considered consistent with the description herein thata holder additionally can be configured to accept a sample, such as in asample tube. Thus, in embodiments described elsewhere herein, a rackaccepts a number of sample tubes and a number of corresponding holdersin such a manner that the sample tubes and holders can be separately andindependently loaded from one another. Nevertheless, in otherembodiments, a holder can be configured to also accept a sample, forexample in a sample tube. And thus, a complementary rack is configuredto accept a number of holders, wherein each holder has a sample as wellas reagents and other items. In such an embodiment, the holder isconfigured so that the sample is accessible to a sample identificationverifier.

Kits

The holder described herein may be provided in a sealed pouch, to reducethe chance of air and moisture coming into contact with the reagents inthe holder. Such a sealed pouch may contain one or more of the holdersdescribed herein, such as 2, 4, 6, 8, 10, 12, 16, 20, or 24 holders.

The holder may also be provided as part of a kit for carrying out samplepreparation, wherein the kit comprises a first pouch containing one ormore of the holders described herein, each of the holders configuredwith liquid reagents for, e.g., lysis, wash, and release, and a secondpouch, having an inert atmosphere inside, and one or more reagent tubescontaining lyophilized PCR reagents, as shown in FIGS. 8A-D. Such a kitmay also be configured to provide for analysis of multiple samples, andcontain sufficient PCR reagents (or other amplification reagents, suchas for RT-PCR, transcription mediated amplification, strand displacementamplification, NASBA, helicase dependent amplification, and otherfamiliar to one of ordinary skill in the art, and others describedherein) to process such samples, and a number of individual holders suchas 2, 4, 6, 8, 10, 12, 16, 20, or 24 holders.

Reagent Tubes

As referenced elsewhere herein, the containers 554 that containlyophilized reagents are 0.3 ml tubes that have been further configuredto have a star-shaped—or stellated—pattern (see FIGS. 4A and 4B) ontheir respective bottom interior surfaces. Still other tubes for useherein, as well as for other uses not herein described, can be similarlyconfigured. Thus, for example, the benefits afforded by the star-shapedpattern also accrue to reagent tubes that contain liquid samples thatare directly pipetted out of the tubes (as well as to those tubes thatinitially hold solids that are constituted into liquid form prior topipetting). Other size tubes that would benefit from such a star-shapedpattern have sizes in the range 0.1 ml to 0.65 ml. for example.

The star-shaped pattern ensures that when a fluid is withdrawn from thetube, a pipette tip can be bottomed out in the tube and still be able towithdraw the entire, or almost the entire fluid from the tube, as shownin FIG. 5. This is important because, when working with such smallvolumes, and when target DNA can be present in very few copies, sampleloss due to imperfections of pipetting is to be minimized to everyextent possible.

The design of the star shaped pattern is important, especially whenusing for recovery of DNA/RNA present in very small numbers in theclinical sample. The stellated pattern should enable pipetting of mostof the liquid (residual volume <1 microliter) when used with a pipettebottomed out with the bottom of the tube. Additionally, the stellatedpattern should be designed to minimize surface area as well as dead-endgrooves that tend to have two undesirable effects—to trap liquid as wellas to increase undesirable retention of polynucleotides by adsorption.

FIG. 5 is now described, as follows. FIG. 5 has a number of panels, A-G,each representing, in sequence, a stage in a pipetting operation. At A,a pipette tip 2210, containing a liquid 2211 (such as a buffersolution), is positioned directly or approximately above the center ofreagent tube 2200. The tube contains a number of lyophilized pellets2212, and is sealed by a layer 2214, such as of foil. The foil may beheat-sealed on to the top of the tube. Although a laminate layer, asfurther described herein, can be placed on the reagent tube, typically alayer of aluminum foil is adequate, where the tube contents are solid,e.g., lyophilized, reagents. In some embodiments, the top of the reagenttube has chamfer edges to reduce expansion of the top rim of the tubeduring heat sealing of a foil on the top of the tube. The tube mayfurther comprise an identifiable code, such as a 1-D or a 2-D bar-codeon the top. Such a code is useful for identifying the composition of thereagents stored within, and/or a batch number for the preparationthereof, and/or an expiry date. The code may be printed on with, forexample, an inkjet or transfer printer.

Stellated pattern 2203 on the bottom interior surface of the tube 2200is shown. At B, the pipette tip is lowered, piercing seal 2214, andbrought into a position above the particles 2212. At C the liquid 2211is discharged from the pipette tip on to the particles, dissolving thesame, as shown at D. After the particles are fully dissolved, forming asolution 2218, the pipette tip is lowered to a position where it is incontact with the stellated pattern 2203. A E, the pipette tip is causedto suck up the solution 2218, and at F, the tip may optionally dischargethe solution back into the tube. Steps E and F may be repeated, asdesired, to facilitate dissolution and mixing of the lyophilizedcomponents into solution. At step G, after sucking up as much of thesolution 2218 as is practicable into the pipette tip, the pipette tip iswithdrawn from the tube. Ideally, 100% by volume of the solution 2218 isdrawn up into the pipette tip at G. In other embodiments, and dependingupon the nature of solution 2218, at least 99% by volume of the solutionis drawn up. In still other embodiments, at least 98%, at least 97%, atleast 96%, at least 95%, and at least 90% by volume of the solution isdrawn up.

The design of the stellated or star-shaped pattern can be optimized tomaximize the flow rate of liquid through the gaps in-between a bottomedout pipette, such as a p1000 pipette, and the star pattern, and isfurther described in U.S. provisional patent application Ser. No.60/959,437, filed Jul. 13, 2007, incorporated herein by reference. Itwould be understood that, although the description herein pertains topipettes and pipette tips typically used in sample preparation ofbiological samples, the principles and detailed aspects of the designare as applicable to other types of pipette and pipette tip, and may beso-adapted.

FIG. 4A shows a cross sectional perspective view of a reagent tube 2200having side wall 2201 and bottom 2202. Interior surface 2204 of thebottom is visible. A star-shaped cutout 2203 is shown in part, as threeapical grooves.

Typically the star-shaped pattern is present as a raised portion on thelower interior surface of the tube. Thus, during manufacture of areagent tube, such as by injection moulding, an outer portion of themould is a cavity defining the exterior shape of the tube. An interiorshape of the tube is formed by a mould positioned concentrically withthe outer portion mould, and having a star-shaped structure milled outof its tip. Thus, when liquid plastic is injected into the space betweenthe two portions of the mould, the star-shape is formed as a raisedportion on the bottom interior surface of the tube.

The exemplary star pattern 2203 shown in FIG. 4B in plan view resemblesa “ship's wheel” and comprises a center 2209, a circular ring 2207centered on center 2209, and 8 radial segments configured as radialgrooves 2205. Each groove meets the other grooves at center 2209, andhas a radial end, also referred to as an apex or vertex. Star pattern2203 has 8 grooves, but it would be understood that a star patternhaving fewer or a greater number of grooves, such as 3, 4, 6, 10, or 12,would be consistent with the design herein. The number of grooves of thestar should be minimum consistent with effective liquid pipetting andalso spaced apart enough not to trap the tip of any of the pipette tipsto be used in the liquid handling applications.

Center 2209 is typically positioned coincidentally with the geometriccenter of the bottom of reagent tube 2200. The tube is typicallycircular in cross-section, so identifying its center (e.g., at acrossing point of two diameters) is normally straightforward. Center2209 may be larger than shown in FIG. 4B, such as may be a circularcutout or raised portion that exceeds in diameter of the region formedby the meeting point of grooves 2205.

Ring 2207 is an optional feature of star-shaped pattern 2203. Typicallyring 2207 is centered about center 2209, and typically it also has adimension that corresponds to the lower surface of a pipette tip. Thus,when a pipette tip ‘bottoms out’ in the bottom of reagent tube 2200, thebottom of the pipette tip rests in contact with ring 2207. Ring 2207 isthus preferably a cut-our or recessed feature that can accommodate thepipette tip and assist in guiding its positioning centrally at thebottom of the tube. In other embodiments more than one, such as 2, 3, or4 concentric rings 2207 are present.

The star pattern is configured to have dimensions that give an optimalflow-rate of liquid out of the reagent tube into a suitably positionedpipette tip. The star pattern is shown in FIG. 4B as being significantlysmaller in diameter than the diameter of the tube at its widest point.The star pattern may have, in various embodiments, a diameter (measuredfrom center 2209 to apex of a groove 2205) from 5-20% of the diameter ofthe reagent tube, or from 10-25% of the diameter of the reagent tube, orfrom 15-30% of the diameter of the reagent tube, or from 20-40% of thediameter of the reagent tube, or from 25-50% of the diameter of thereagent tube, or from 30-50% the diameter of the reagent tube, or from40-60% the diameter of the reagent tube, or from 50-75% the diameter ofthe reagent tube, or from 65-90% the diameter of the reagent tube.

The grooves 2205 are thus separated by ridges (occupying the space inbetween adjacent grooves). In the embodiment shown, the grooves arenarrower (occupy a smaller radial angle) than the gaps between them. Inother embodiments, the grooves may be proportionately wider than thegaps between them. In such embodiments, it may be more appropriate todescribe them as having ridges instead of grooves. In other embodiments,the grooves and ridges that separate them are of equal widths at eachradial distance from the center.

The grooves that form the apices of the star may be rounded in theirlower surfaces, such as semi-circular in cross section, but aretypically V-shaped. They may also be trapezoid in cross-section, such ashaving a wider upper portion than the bottom, which is flat, the upperportion and the bottom being connected by sloping walls.

In some embodiments, for ease of manufacture, the grooves end on thesame level in the bottom of the tube. Thus the radial ends are alldisposed on the circumference of a circle. In other embodiments, thegrooves do not all end on the same level. For example, grooves mayalternately end on different levels, and thus the ends are alternatelydisposed on the respective circumferences of two circles that occupydifferent planes in space from one another.

Grooves 2205 are shown in FIG. 4B as having equal lengths (as measuredfrom center 2209 to apex). This need not be so. In alternativeembodiments, grooves may have different lengths from one another, forexample, as alternating lengths on alternating grooves, where there arean even number of grooves. Furthermore, apices may be rounded, ratherthan pointed.

Typically the grooves taper uniformly in width and depth from center2209 to each respective apex. Still other configurations are possible,such as a groove that follows a constant width, or depth, out to aparticular radial extent, such as 30-60% of its length, and then narrowsor becomes shallower towards its apex. Alternatively, a groove may startnarrow at center 2209, widen to a widest region near its midpoint oflength, and then narrow towards its apex. Still other possibilities, notdescribed herein, are consistent with the stellated pattern.

In a 0.3 ml tube, the width of each groove 2205 at its widest point istypically around 50 microns, and the width typically tapers uniformlyfrom a widest point, closest to or at center 2209, to the apex.

In a 0.3 ml tube, the depth of a groove at the deepest point istypically around 25-50 microns and the depth typically tapers uniformlyfrom a deepest point, closest to or at center 2209, to an apex.

In a 0.3 ml tube, the radius of the star formed from the grooves,measured as the shortest distance from center 2209 to apex, is typicallyaround 0.5 mm, but may be from 0.1-1 mm, or from 0.3-2 mm.

In another embodiment, in a 0.3 ml tube, the grooves should be roundedoff and less than 100 microns deep, or less than 50 microns deep, orless than 25 microns deep.

The stellated pattern typically has a rotation axis of symmetry, theaxis disposed perpendicular to the bottom of the tube and through center2209, so that the grooves are disposed symmetrically about the rotationaxis. By this is meant that, for n grooves, a rotation of 2π/n about thecentral (rotational) axis can bring each groove into coincidence withthe groove adjacent to it.

The stellated shape shown in FIG. 4B is not limiting in that itcomprises a number of radially disposed grooves 2205, and an optionalcircular ring 2207. Other star-shaped geometries may be used, and,depending upon ease of manufacture, may be preferred. For example, astar can be created simply be superimposing two or more polygons havinga common center, but offset rotationally with respect to one anotherabout the central axis. (See, for example “star polygons” described athttp://mathworld.wolfram.com/StarPolygon.html.) Such alternative mannersof creating star-shaped patterns are utilizable herein.

Rack

The holders herein are typically configured to fit into a dedicatedrack, the rack being configured to hold one or more such holders, eitheron a laboratory benchtop, or situated in a dedicated apparatus havingone or more other functions, such as automated pipetting. Twoperspective views of an exemplary rack 800, configured to accept 12sample tubes and 12 holders, are shown in FIG. 9.

For example, a rack may accept 2, 4, 6, 8, 10, 12, 16, or 20 samples, insample tubes 802, and corresponding holders 804. In certain embodimentsthe holders each slide into, and lock into, place in the rack, such aswith a cam locking mechanism that is recognized as locked audibly and/orphysically, or such as with a mechanical key. The rack can be configuredso that the holders, when positioned in it, are aligned for properpipette tip pick-up using an automated liquid dispenser as furtherdescribed herein.

In certain embodiments, the rack is configured to accept the samples inindividual sample tubes 802, each mounted adjacent to a correspondingholder.

The rack can be designed so that it can be easily removed from adedicated apparatus and carried between the laboratory environmentexternal to the apparatus, such as a bench, and the apparatus. Incertain embodiments, the rack is designed to be stable on a horizontalsurface, and not easily toppled over during carriage. In certainembodiments the rack has a handle 806 to ease lifting and moving, andthe handle can be locked into a vertical position, during carriage, alsoto reduce risk of the rack being toppled over. The handle has a softgrip 808 in its middle, and is attached to a metallic base member 810having 4 feet that also serve as position locators when inserting therack into a dedicated apparatus. The portion of the rack 812 thataccepts the samples and holders can be made of plastic, and comprises 12slots, and may be disposable.

In certain embodiments, the interior of the rack around the location ofprocess tubes in the various holders is configured to have clearance fora heater assembly and/or a magnetic separator as further describedherein. For example, the rack is configured so that process tubes on theindividual holders are accepted by heater units in a heater assembly asfurther described in U.S. provisional patent application Ser. No.______, filed Jul. 13, 2008.

Still other embodiments of a rack are described in U.S. patentapplication Ser. No. ______, filed Jul. ______, 2008, and incorporatedherein by reference.

Liquid Dispenser

Additionally, the holders herein are configured to accept pipetteoperations both performed manually by an operator, and by an automatedpipette head. An exemplary automated pipette head is described in U.S.provisional application Ser. No. 60/959,437, filed Jul. 13, 2008, andincorporated herein by reference in its entirety. Typical features of anautomated pipette head suitable for operating with holders as describedherein include at least: an ability to pick up pipette tips from the oneor more sockets, and to return pipette tips to such sockets after use;to strip and discard a pipette tip from a pipette head after use or uponencountering an error; move a pipette tip with precision from onelocation of a given holder to another so that, for example, liquidreagents can be located and added to solid reagents to make upsolutions, and various liquid reagents can be mixed with one anotherduring a sample preparation protocol. Furthermore, it is desirable thatsuch an automated pipette device can operate on several, such as 2, 3,4, or 6, holders simultaneously, and thereby perform certain operationsin parallel. Thus the pipette head should move in three degrees offreedom.

Heater Assembly & Magnetic Separator

Furthermore, the holders herein are configured so that the process tubeis heated by a dedicated heating unit 1001, for example situated in anapparatus for carrying out sample preparation on multiple samples inparallel, as shown in FIG. 10. Typically such a heater assemblycomprises one or more independently controllable heater units 1010, eachof which comprises a heat block configured to heat a process tube in aholder. In one embodiment, a heat element is a power resistor. The righthand panel of FIG. 10 shows how holders loaded in a rack can bepositioned in close proximity to a dedicated heating unit.

Yet additionally, the holders herein are configured so that the processtube is in close enough proximity to a magnetic assembly that separationof magnetic particles from reagents in solution in the process tube canbe accomplished. An exemplary magnetic separator is configured to moveone or more magnets relative to the one or more process tubes.Typically, the magnet is mounted in such a way that it can be moved inproximity to the process tubes, either in an automated fashion such asunder control of a processor, or manually. The magnet can be made ofneodymium (e.g., from K &J Magnetics, Inc.) and can have a magneticstrength of 5,000-15000 Gauss (Brmax). The poles of the magnets can bearranged such that one pole faces the heat blocks and the other facesaway from the heat blocks.

Advantageously, the heater assembly and magnetic separator operatetogether to permit successive heating and separation operations to beperformed on liquid materials in the one or more process tubes withouttransporting either the liquid materials or the process tubes todifferent locations to perform either heating or separation. Anexemplary heater assembly and magnetic separator are further describedin U.S. provisional patent application Ser. No. 60/959,437, filed Jul.13, 2008, and incorporated herein by reference in its entirety.

Apparatus Overview

A schematic overview of an apparatus 981 for carrying out automatedsample preparation on multiple samples in parallel, according to stepsexemplified elsewhere herein, is shown in FIG. 11. The geometricarrangement of the components of system 981 is exemplary and notintended to be limiting.

A processor 980, such as a microprocessor, is configured to controlfunctions of various components of the system as shown, and is therebyin communication with each such component requiring control. It is to beunderstood that many such control functions can optionally be carriedout manually, and not under control of the processor. Furthermore, theorder in which the various functions are described, in the following, isnot limiting upon the order in which the processor executes instructionswhen the apparatus is operating. Thus, processor 980 can be configuredto receive data about a sample to be analyzed, e.g., from a samplereader 990, which may be a barcode reader, an optical character reader,or an RFID scanner (radio frequency tag reader). Processor 980 can beconfigured to accept user instructions from an input device 984, wheresuch instructions may include instructions to start analyzing thesample, and choices of operating conditions. Processor 980 can be alsoconfigured to communicate with a display 982, so that, for example,information about an analysis is transmitted to the display and therebycommunicated to a user of the system. Such information includes but isnot limited to: the current status of the apparatus; progress of PCRthermocycling; and a warning message in case of malfunction of eithersystem or cartridge. Additionally, processor 980 may transmit one ormore questions to be displayed on display 982 that prompt a user toprovide input in response thereto. Thus, in certain embodiments, input984 and display 982 are integrated with one another. Processor 980 canbe optionally further configured to transmit results of an analysis toan output device 986 such as a printer, a visual display, a display thatutilizes a holographic projection, or a speaker, or a combinationthereof. Processor 980 can be still further optionally connected via acommunication interface such as a network interface to a computernetwork 988.

Processor 980 can be further configured to control various aspects ofsample preparation and diagnosis, as follows in overview. In FIGS. 1Aand 1B, the apparatus 981 is configured to operate in conjunction with acomplementary rack 970. The rack is itself configured, as furtherdescribed herein, to receive a number of biological samples 996 in aform suitable for work-up and diagnostic analysis, and a number ofholders 972—as further described herein, such as in connection withFIGS. 1A, 1B, 2, and 3A-C, that are equipped with various reagents,pipette tips and receptacles. The rack is configured so that, duringsample work-up, samples are processed in the respective holders, theprocessing including being subjected, individually, to heating andcooling via heater assembly 977.

The heating functions of the heater assembly can be controlled by theprocessor 980. Heater assembly 977 operates in conjunction with aseparator 978, such as a magnetic separator, that also can be controlledby processor 980 to move into and out of close proximity to one or moreprocessing chambers associated with the holders 972, wherein particlessuch as magnetic particles are present.

Liquid dispenser 976, which similarly can be controlled by processor980, is configured to carry out various suck and dispense operations onrespective sample, fluids and reagents in the holders 972, to achieveextraction of nucleic acid from the samples. Liquid dispenser 976 cancarry out such operations on multiple holders simultaneously. Samplereader 990 is configured to transmit identifying indicia about thesample, and in some instances the holder, to processor 980. In someembodiments a sample reader is attached to the liquid dispenser and canthereby read indicia about a sample above which the liquid dispenser issituated. In other embodiments the sample reader is not attached to theliquid dispenser and is independently movable, under control of theprocessor. Liquid dispenser 976 is also configured to take aliquots offluid containing nucleic acid extracted from one or more samples anddirect them to storage area 974, which may be a cooler. Area 974contains, for example, a PCR tube corresponding to each sample.

Embodiments of the apparatus shown in outline in FIG. 11, as with otherexemplary embodiments described herein, are advantageous because they donot require locations within the apparatus suitably configured forstorage of reagents. Therefore, the apparatus in FIG. 11 isself-contained and operates in conjunction with holders 972, wherein theholders are pre-packaged with reagents, such as in locations within itdedicated to reagent storage.

The apparatus of FIG. 11 may be configured to carry out operation in asingle location, such as a laboratory setting, or may be portable sothat they can accompany, e.g., a physician, or other healthcareprofessional, who may visit patients at different locations. Theapparatus is typically provided with a power-cord so that they canaccept AC power from a mains supply or generator. The apparatus may alsobe configured to operate by using one or more batteries and therefore isalso typically equipped with a battery recharging system, and variouswarning devices that alert a user if battery power is becoming too lowto reliably initiate or complete a diagnostic analysis.

The apparatus of FIG. 11 may further be configured, in otherembodiments, for multiplexed sample analysis and/or analysis of multiplebatches of samples, where, e.g., a single rack holds a single batch ofsamples. Each component shown in FIG. 11 may therefore be present asmany times as there are batches of samples, though the variouscomponents may be configured in a common housing.

EXAMPLES Example 1 Reagent Holder

An exemplary reagent holder consistent with the description herein hasthe following dimensions and capacities:

-   -   180 mm long×22 mm wide×100 mm tall;    -   Made from Polypropylene.    -   One snapped-in low binding 1.7 ml tube that functions as a        process tube.    -   3 built-in tubes that function as receptacles for reagents, as        follows:        -   One tube containing 200-1000 μl of wash buffer (0.1 mM Tris,            pH 8).        -   One tube containing 200-1000 μl of release solution (40 mM            NaOH).        -   One tube containing 200-1000 μl of neutralization solution            (330 mM Tris, pH 8.0).    -   One built-in tube that functions as a waste chamber (will hold        ˜4 ml of liquid waste).    -   3 receptacles to accept containers for solid reagents. Snap-in        0.3 ml or 0.65 ml PCR tubes (which are typically stored        separately from the reagent holder) are placed in each of these        locations, and contain, respectively:        -   lyophilized sample preparation reagents (lysis enzyme mix            and magnetic affinity beads).        -   First lyophilized PCR master mix, probes and primers for a            first target analyte detection.        -   Second lyophilized PCR master mix, probes and primers for a            second target analyte detection (only offered in select            cases, such as detection of Chlamydia and Gonorrhea from            urine).    -   4 pipette tips located in 4 respective sockets.    -   Pipette tip Sheath: The pipette tips have a sheath/drip tray        underneath to help capture any drip from the pipette tips after        being used, and also to prevent unwanted contamination of the        instrument.    -   Handle and Flex-Lock allows easy insertion, removal, and        positive location of strip in rack.    -   One or more labels: positioned upward facing to facilitate ease        of reading by eye and/or, e.g., a bar-code reader, the one or        more labels containing human and machine readable information        pertaining to the analysis to be performed.

It is to be understood that these dimensions are exemplary. However, itis particularly desirable to ensure that a holder does not exceed thesedimensions so that a rack and an apparatus that accommodates the reagentholder(s) does not become inconveniently large, and can be suitablysituated in a laboratory, e.g., on a bench-top.

Example 2 Disposable Reagent Holder Manufacturing

Simple fixtures can be designed and machined to enable handling andprocessing of multiple strips. There are five steps that can beperformed to produce this component. The disposable reagent holder willbe placed in a fixture and filled with liquids usingmanual/electric-multiple pipetting. Immediately after dispensing allliquids into the strip, foil will be heat sealed to the plastic usingexemplary heat seal equipment (Hix FH-3000-D Flat Head Press) and thefoil trimmed as required. After heat sealing liquids on board, allpellets in tubes can be snapped into the strip, pipette tips can beinserted in their respective sockets, and a barcode label can beaffixed. Desiccant packs can be placed into the blow molded orthermoformed rack designed to house 12 holders. Twelve disposable stripswill be loaded into the rack and then sealed with foil. The sealed bagwill be placed into a carton and labeled for shipping.

Example 3 Foil-Sealing of Buffer Containing Reagent Tubes

Tubes containing buffers have to be sealed with high moisture vaporbarrier materials in order to retain the liquid over a long period oftime. Disposable holders may need to have a shelf life of 1-2 years, andas such, they should not lose more than say 10-15% of the liquid volumeover the time period, to maintain required volume of liquid, and tomaintain the concentration of various molecules present in the solution.Moreover, the materials used for construction of the tube as well as thesealing laminate should not react with the liquid buffer. Specialplastic laminates may provide the moisture barrier but they may have tobe very thick (more than 300 μm thick), causing the piercing force to goup tremendously, or of special, expensive polymer (such as Aclar).Aluminum foils, even a thin foil of a few hundred angstrom provides aneffective moisture barrier but bare aluminum reacts with some liquidbuffers, such as sodium hydroxide, even an aluminum foil with a sprayedcoating of a non-reactive polymer may not be able to withstand thecorrosive vapors over a long time. They may react through tiny pin holespresent in the coating and may fail as a barrier over time.

For these reasons, aluminum foils with a laminate structure have beenidentified as a suitable barrier, exemplary properties of which aredescribed below:

1. Sealing

-   -   Heat seals to unitized polypropylene strip (sealing temp        ˜170-180° C.)    -   No wrinkling, cracking and crazing of the foil after sealing

2. Moisture Vapor Transmission Rate (MVTR)

-   -   Loss of less than 10% liquid (20 microliters from a volume of        200 microliter) for a period of 1 year stored at ambient        temperature and pressure. (effective area of transport is ˜63        mm²); Approximate MVTR 0.8 cc/m²/day

3. Chemistry

-   -   Ability to not react with 40 mM Sodium Hydroxide (pH<12.6): foil        should have a plastic laminate at least 15 microns thick closer        to the sealed fluid.    -   Ability to not react with other buffers containing mild        detergents

4. Puncture

-   -   Ability to puncture using a p1000 pipette with a force less than        3 lb    -   Before puncturing, a fully supported membrane 8 mm in diameter        will not stretch more than 5 mm in the orthogonal direction    -   After puncturing, the foil should not seal the pipette tip        around the circumference of the pipette.

5. Other Features

-   -   Pin-hole free    -   No bubbles in case of multi-laminate structures.

Example 4 Mechanism of Piercing Through a Plasticized Laminate andWithdrawing Liquid Buffer

The aluminum laminate containing a plastic film described elsewhereherein serves well for not reacting with corrosive reagents such asbuffers containing NaOH, and having the favorable properties ofpierceability and acting as a moisture barrier. However, it presentssome additional difficulties during piercing. The aluminum foil tends toburst into an irregular polygonal pattern bigger than the diameter ofthe pipette, whereas the plastic film tends to wrap around the pipettetip with minimal gap between the pipette and the plastic film. Thediameter of the hole in the plastic film is similar to the maximumdiameter of the pipette that had crossed through the laminate. Thiswrapping of the pipette causes difficulty in dispensing and pipettingoperations unless there is a vent hole allowing pressures to equilibratebetween outside of the tube and the air inside of the tube.

A strategy for successful pipetting of fluid is as follows:

-   -   1. Pierce through the laminate structure and have the pipette go        close to the bottom of the reagent tube so that the hole created        in the laminate is almost as big as the maximum diameter of the        pipette (e.g., ˜6 mm for a p1000 pipette)    -   2. Withdraw the pipette up a short distance so that a small        annular vent hole is left between the pipette and the laminate.        The p1000 pipette has a smallest outer diameter of 1 mm and        maximum outer diameter of 6 mm and the conical section of the        pipette is about 28 mm long. A vent hole thickness of a hundred        microns is enough to create a reliable vent hole. This        corresponds to the pipette inserted to a diameter of 5.8 mm,        leaving an annulus of 0.1 mm around it.    -   3. Withdraw fluid from the tube. Note that the tube is designed        to hold more fluid than is necessary to withdraw from it for a        sample preparation procedure.

Example 5 Foil Piercing and Dissolution of Lyophilized Reagents

The containers of lyophilized reagents provided in conjunction with aholder as described herein are typically sealed by a non-plasticizedaluminum foil (i.e., not a laminate as is used to seal the reagenttubes). Aluminum foil bursts into an irregular polygonal pattern whenpierced through a pipette and leaves an air vent even though the pipetteis moved to the bottom of the tube. In order to save on reagents, it isdesirable to dissolve the reagents and maximize the amount withdrawnfrom the tube. To accomplish this, a star-ridged (stellated) pattern isplaced at the bottom of the container to maximize liquid volumewithdrawn, and flow velocity in between the ridges.

Exemplary steps for dissolving and withdrawing fluid are as follows:

-   -   1. Pierce through the pipette and dispense the fluid away from        the lyophilized material. If the pipette goes below the level of        the lyophilized material, it will go into the pipette and may        cause jamming of the liquid flow out of the pipette.    -   2. Let the lyophilized material dissolve for a few seconds.    -   3. Move pipette down touching the ridged-bottom of the tube    -   4. Perform an adequate number of suck and spit operations (4-10)        to thoroughly mix the reagents with the liquid buffer.    -   5. Withdraw all the reagents and move pipette to dispense it        into the next processing tube.

Example 6 Material and Surface Property of the Lysis Tube

The material, surface properties, surface finish has a profound impacton the sensitivity of the assay performed. In clinical applications,DNA/RNA as low as 50 copies/sample (˜1 ml volume) need to be positivelydetected in a background of billions of other molecules, some of whichstrongly inhibit PCR. In order to achieve these high level ofsensitivities, the surface of the reaction tube as well as the materialof the surface has to be chosen to have minimal binding ofpolynucleotides. During the creation of the injection molding tool tocreate these plastic tubes, the inherent surfaces created by machiningmay have large surface area due to cutting marks as large as tens ofmicrons of peaks and valleys. These surfaces have to be polished to SPIA1/A2 finish (mirror finish) to remove the microscopic surfaceirregularities. Moreover, the presence of these microscopic valleys willtrap magnetic beads (0.5-2μ) at unintended places and cause irregularperformance. In addition to actual surface roughness, the surfacehydrophobicity/surface molecules present may cause polynucleotides tostick at unintended places and reduce sensitivity of the overall test.In addition to the base material uses, such as homogenous polupropyleneand other polymers, specific materials used during the molding of thesetubes, such as mold release compounds or any additives to aid in thefabrication can have a profound impact on the performance of thereactions.

The foregoing description is intended to illustrate various aspects ofthe present inventions. It is not intended that the examples presentedherein limit the scope of the present inventions. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A reagent holder comprising: a connecting member having an upper sideand, opposed to the upper side, an underside; a process tube affixed tothe connecting member and having an aperture located in the connectingmember; at least one socket, located in the connecting member, thesocket configured to accept a pipette tip; two or more reagent tubesdisposed on the underside of the connecting member, each of the reagenttubes having an inlet aperture located in the connecting member; and oneor more receptacles, located in the connecting member, wherein the oneor more receptacles are each configured to receive a reagent tubeinserted from the upper side of the connecting member.
 2. The holder ofclaim 1, configured for a single use only.
 3. The holder of claim 1,additionally comprising: a waste chamber disposed on the underside ofthe connecting member and having an inlet aperture located in theconnecting member.
 4. (canceled)
 5. The holder of claim 1, additionallycomprising: a pipette tip sheath removably affixed to an underside ofthe connecting member, the sheath configured to surround the at leastone socket and a tip and lower portion of a pipette tip when the pipettetip is stationed in the at least one socket. 6-8. (canceled)
 9. Theholder of claim 1, wherein the connecting member further comprises amechanical key that ensures that the holder is accepted by acomplementary member in a supporting rack.
 10. The holder of claim 1,wherein at least the connecting member is made of a flexible material sothat the holder can be reversibly snapped into a complementary member ina supporting rack. 11-12. (canceled)
 13. The holder of claim 1, whereinone or more of the two or more reagent tubes each contains liquidreagents.
 14. The holder of claim 13, wherein the liquid reagents areselected from the group consisting of: PCR reagents in solution, andsample preparation reagents. 15-16. (canceled)
 17. The holder of claim1, further comprising a laminate structure covering the apertures of thetwo or more reagent tubes.
 18. The holder of claim 17, wherein thelaminate comprises, in sequence: a layer of aluminum foil, an adhesivelayer, a polymer layer, and a heat seal layer, wherein the heat seallayer is adjacent the one or more reagent tubes.
 19. The holder of claim18, wherein the aluminum foil is 0.1-15 microns thick, and the polymerlayer is 15-25 microns thick.
 20. The holder of claim 18, wherein thealuminum is 0.1-1 microns thick, and the polymer layer is 25-30 micronsthick.
 21. The holder of claim 18, wherein the aluminum layer is a layerof evaporated or sputtered aluminum.
 22. The holder of claim 18, whereinthe polymer is polypropylene.
 23. The holder of claim 18, furthercomprising a raised portion on the upper side of the connecting member,and surrounding the inlet apertures of the two or more reagent tubes,wherein the laminate is attached to the raised portion.
 24. The holderof claim 1, wherein the process tube has a stellated ridge-pattern onits interior bottom surface.
 25. (canceled)
 26. The holder of claim 1,wherein the connecting member, process tube, and the two or more reagenttubes are made from a single piece.
 27. The holder of claim 3, whereinthe connecting member, process tube, the two or more reagent tubes, andthe waste chamber are made from a single piece.
 28. The holder of claim1, wherein the connecting member, process tube, and the two or morereagent tubes are made of polypropylene.
 29. The holder of claim 3,wherein the connecting member, process tube, two or more reagent tubes,and the waste chamber are made of polypropylene.
 30. (canceled)
 31. Theholder of claim 1, wherein the various inlet apertures are configured toaccept a pipette tip. 32-33. (canceled)
 34. A kit comprising: a firstpouch containing one or more of the holders of claim 1; and a secondpouch, having an inert atmosphere inside, and one or more tubescontaining lyophilized PCR reagents.
 35. (canceled)
 36. A unitizedreagent holder, comprising: a strip, to which is attached: a singleprocess tube; one or more containers, each of which holding a reagentselected from the group consisting of: a sample preparation reagent, PCRreagents for a first analyte, and one or more liquid reagents; and oneor more sockets configured to hold one or more pipette tips. 37-39.(canceled)
 40. The unitized reagent holder of claim 36, furthercomprising a laminate that seals closed an inlet of each of the one ormore receptacles.
 41. The unitized reagent holder of claim 35, whereinat least one of the one or more receptacles is removable.
 42. Theunitized reagent holder of claim 35, further comprising a pipette tipsheath configured to surround the one or more pipette tips.
 43. Theunitized reagent holder of claim 35, further comprising a waste tube.44. A reagent holder comprising: a process tube; one or more pipettetips; and one or more reagent tubes, wherein the one or more reagenttubes contain, respectively, sufficient quantities of one or morereagents for carrying out extraction of polynucleotides from a sample,and wherein the process tube, the one or more pipette tips, and the oneor more reagent tubes are each joined to a single connecting member. 45.The holder of claim 44, further comprising: a waste tube; and a pipettetip sheath configured to surround the one or more pipette tips.
 46. Theholder of claim 1, wherein the connecting member has a first end and asecond end, and the process tube is located at either of the first orsecond ends. 47-66. (canceled)